Studies of DNA replication of plasmid ColEl have been continued. Transcripts (RNA II) that start 555 nucleotides upstream of the replication origin by RNA polymerase form a hybrid with the template DNA. The hybridized transcript is cleaved by ribonuclease H at the origin and used as the primer for leading strand synthesis by DNA polymerase I. ColEl DNA can replicate also in the absence of the RNase H and DNA polymerase I. RNA Il hybridized with the template DNA displaces the nontranscribed strand on which lagging strand synthesis takes place. RNA II that extends beyond the normal replication origin terminates at various positions. When a stretch of dA residues is inserted at various positions of the template strand downstream of the origin most transcripts that hybridize with the template DNA terminates at the stretch. When RNA II transcription templates at a dA stretch, a single-stranded region of a defined length is formed on the nontranscribed strand. For initiating of synthesis of the lagging strand, formation of a single-stranded legion of at least 45 nucleotides long is required. The electron microscopic studies show that the host Dna B protein with a helicase activity binds to this short single-stranded region and unwinds the template DNA to allow assembly of a replisome for lagging strand synthesis.